The goal of this grant is to examine B cell ontogeny and development of the antibody repertoire in rabbits. We have recently proposed a model for B cell development that includes a major role for gut-associated lymphoid tissue (GALT). In the model, all B cells develop early in ontogeny and during neonatal life, and because they utilize a limited number of VDJ genes with no somatic mutation, they comprise a limited antibody repertoire, designated the neonatal repertoire. These neonatal rabbits are relatively immunoincompetent. Further, the model proposes that shortly after birth the B cells migrate to GALT and the VDJ genes diversify by somatic gene conversion, resulting in the primary antibody repertoire that makes the rabbit immunocompetent. Finally, the model suggests that the repertoire is maintained by self-renewing CD5+ B cells. The experiments designed to test this model are part of three specific aims: 1) To determine the extent to which B-lymphopoiesis occurs in adult rabbits, 2) To determine if GALT is the site of development of the primary antibody repertoire, and 3) To determine whether microbial antigens are required to generate diversified VDJ genes. The experiments include developing monoclonal antibodies that can be used to identify pre-B cells, searching for V-D and D4 heavy chain gene rearrangements by analyzing intrachromosomally-derived circular DNA, developing Rb-SCID mice to determine if rabbit B cells are self-renewing, examining VDJ genes of germfree rabbits and rabbits with GALT surgically removed as neonates to determine if they remain undiversified, searching for on- going VDJ gene diversification in GALT, and testing whether undiversified VDJ genes bind microbial antigens from GALT. This work is important because the rabbit B cell system is different from that of all other animals studied thus far and the results will likely lead to new insights into mechanisms by which the humoral immune system functions. Especially important are the studies with gut-associated lymphoid tissue (GALT). GALT is the largest immunologic organ in the body and yet relatively little is known of its function. Results from work proposed in this grant should help to elucidate how GALT contributes to immune functions.